Tissue Factor Antibody Research Articles

Tissue factor: A link between metastatic colorectal cancer and thrombosis in patients in the CALGB (Alliance)/SWOG 80405 trial.

198 Background: Tissue factor (TF) is a transmembrane protein that plays a crucial role in thrombosis. TF expression is elevated in various types of cancer, including colorectal cancer (CRC) and is associated with more aggressive disease and poor prognosis. The aim of this study was to evaluate TF expression in patients (pts) with metastatic CRC (mCRC) enrolled in the CALGB (Alliance)/SWOG 80405 clinical trial. Understanding the relationship between TF expression and mCRC will provide insights into the role of coagulation in the progression and prognosis of this disease and may have therapeutic implications, particularly in the setting of recent approval of a TF antibody drug conjugate for metastatic cervical cancer and other agents in the pipeline. Methods: 433 pts with mCRC treated with bevacizumab (Bev, n = 226) or cetuximab (Cet, n =207) in combination with first-line chemotherapy in CALGB/SWOG 80405 were included in the analysis. TF RNA was isolated from FFPE tumor samples and sequenced on the HiSeq 2500 (Illumina). Overall survival (OS) and progression-free survival (PFS) were compared between groups of pts categorized by tertiles of TF expression: high (H), medium (M) and low (L). Sensitivity analyses were conducted after stratifying by side. Logrank P-values describe differences without adjustment for pt characteristics. Transcriptome-wide gene association analysis was performed using linear regression, adjusting for age, sex, ethnicity, ECOG PS, location, number of metastases, KRAS, MSI status, treatment with FOLFOX or FOLFIRI, and the first 3 principal components from the RNA-seq data. Gene Ontology enrichment analysis was conducted with the top 100 TF-associated genes. Results: TF expression is associated with genes related to maintenance of epithelium, cell adhesion, and migration, immunoregulatory cytokine production, and metabolic, HER2, and MAP/Kinase pathways. TF-L showed significantly longer median (m)PFS (13.4 vs 10.1 vs 9.2 months (mo), p=0.0022) and mOS (35.4 vs 30.9 vs 25.2, p=0.0051) in the entire cohort. In Cet treated pts, TF-L had significantly longer mOS (41.3 vs 29.9 vs 26.1 mo, p=0.0013). In pts treated with FOLFOX, TF-L had significantly longer PFS (13.2 vs 10.9 vs 8.2 mo, p=0.0014) and OS (33.4 vs 30.9 vs 22.4 mo, p=0.0044). Pts with liver metastases and TF-H had worse PFS (9.2 vs 9.9 vs 13.5 mo, p=0.006) and OS (23.6 vs 29.9 vs 35.1 mo, p=0.016). Conclusions: Our results suggest that pts with TF-L tumors with liver metastases have improved prognosis compared to pts with TF-H and may benefit from FOLFOX and Cet-based treatment. Findings provide rationale for further evaluation of TF as a predictive biomarker and potential therapeutic target in CRC. Trial Identifier NCT00265850. Support: P30CA014089, U10CA180821, U10CA180882, U10CA180820 and UG1CA233277; UG1CA180830, U10CA180888; Pfizer, Genentech. https://acknowledgments.alliancefound.org.

A review of the novel tissue factor antibody-drug conjugate: Tisotumab vedotin.

To review and compare the pharmacology, efficacy, and safety of the novel tissue factor antibody-drug conjugate, tisotumab vedotin. Literature search was performed through PubMed MEDLINE, Google Scholar, ClinicalTrials.gov, and the Food and Drug Administration. Tisotumab vedotin, a novel tissue factor antibody-drug conjugate, was granted accelerated approval by the US FDA on 20 September 2021 for adult patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy. Tisotumab vedotin demonstrated clinical efficacy in a number of solid tumors in innovaTV 201 and more specifically in cervical cancer in the pivotal phase 2 innovaTV 204. In the single-arm innovaTV 204 study, 101 patients with recurrent or metastatic cervical cancer received intravenous tisotumab vedotin at the recommended dose of 2 mg/kg every 3 weeks until disease progression or unacceptable toxicity. The independent review committee confirmed an objective response rate of 24% with 7% complete responses and 17% partial responses. Tisotumab vedotin is associated with several notable adverse events with data from innovaTV 204 including ocular toxicity, hemorrhage, and peripheral neuropathy. Ninety-two percent of patients experienced treatment-related adverse events with 28% experiencing an adverse event of grade 3 or higher. Metastatic cervical cancer has a high risk of relapse with few effective second-line therapeutic options. Current guidelines recommend single agent tisotumab vedotin as a possible option. Ongoing trials will further define its place in therapy.

IL-33 / ST2 Signaling Promotes TF Expression by Regulating NF-κB Activation in Coronary Artery Endothelial Microparticles

IntroductionInterleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial Microparticles(EMPs). Tissue factor (TF) plays a central role in hemostasis and thrombosis.Material and methodsThe study analyzed the coronary blood of level of CD31+EMPs, TF protein and IL-33 protein in Acute Myocardial Infarction (AMI) and stable coronary artery disease (SCAD) patients. Human coronary artery endothelial cells (HCAECs) were treated with IL-33 to obtain EMPs. The TF activity of EMPs was tested by Thermo Fisher by adding the TF antibody. Furthermore, TF and Tissue Factor Pathway Inhibitor (TFPI) protein were tested by ELISA. Finally, NF-κB inhibitor dimethyl fumarate (DMF) and soluble extracellular domain of ST2 coupled to the Fc fragment of human IgG1 (sST2) were added to HCAECs which were treated with IL-33, and the TF protein level was also tested by ELISA.ResultsThe AMI patients had higher level of CD31+EMPs, TF protein and IL-33 protein than the SCAD patients in coronary artery. In AMI patients (N=27), the IL-33 protein positively correlated with CD31+EMPs (r=0.794, p<0.01). According to the ROC curve analysis, the AUC of CD31+EMPs, TF protein and IL-33 protein were 0.888, 0.962 and 0.778 respectively. In the cell culture, the TF activity and TF protein in EMPs increased gradually with time of intervention by the treatment of IL-33. IL-33 binding to the ST2 receptor promoted TF expression by regulating NF-κB activation in EMPs of HCAECs.ConclusionsActivated endothelial cells and EMPs they released simultaneously express TF, which is a risk factor for cardiovascular disease.

Phosphatidylserine-exposing tumor-derived microparticles exacerbate coagulation and cancer cell transendothelial migration in triple-negative breast cancer.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations.Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay.Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells.Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Tissue factor potentiates adherence of breast cancer cells to human umbilical vein endothelial cells under static and flow conditions

ABSTRACT Tissue factor (TF) has been extensively studied for tumor metastasis, but its role in mediating cancer cell adhesion to vasculature remains unknown. This study aimed to measure the ability of TF to mediate the adhesion of breast cancer cells to human umbilical vein endothelial cells (HUVECs). MDA-MB-231 cells expressed the highest TF level and adhered more to HUVECs under static and flow conditions, a neutralizing TF antibody abolished the enhanced adhesion of MDA-MB-231 cells to HUVECs. Recombinant human soluble TF (rTF) bonded β1integrin on HUVECs surfaces, β1 or α3integrin antibody combined with TF antibody abolished more cell-cell adhesion. These data suggested that TF mediated adhesion of breast cancer cells to endothelial cells may rely on β1integrin on HUVECs surfaces.

Evaluation of different commercial antibodies for their ability to detect human and mouse tissue factor by western blotting

BackgroundWestern blotting is used to measure protein expression in cells and tissues. Appropriate interpretation of resulting data is contingent upon antibody validation. ObjectivesWe assessed several commercial anti‐human and anti‐mouse tissue factor (TF) antibodies for their ability to detect TF by western blotting. Material and MethodsWe used human pancreatic cancer cell lines expressing different levels of TF and a mouse pancreatic cancer cell line expressing TF with a matched knockout derivative. ResultsHuman and mouse TF protein detected by western blotting correlated with levels of TF mRNA in these cell lines. The apparent molecular weight of TF is increased by N‐linked glycosylation and, as expected, deglycosylation decreased the size of TF based on western blotting. We found that four commercial anti‐human TF antibodies detected TF in a TF‐positive cell line HPAF‐II whereas no signal was observed in a TF‐negative cell line MIA PaCa‐2. More variability was observed in detecting mouse TF. Two anti‐mouse TF antibodies detected mouse TF in a TF‐positive cell line and no signal was observed in a TF knockout cell line. However, a third anti‐mouse TF antibody detected a nonspecific protein in both the mouse TF‐positive and TF‐negative cell lines. Two anti‐human TF antibodies that are claimed to cross react with mouse TF either recognized a nonspecific band or did not detect mouse TF. DiscussionOur results indicate that there is a range in quality of commercial anti‐TF antibodies. ConclusionWe recommend that all commercial antibodies should be validated to ensure that they detect TF.

A Lipid Peroxidation Product, 4-Hydroxy-2-Nonenal, Triggers Intravascular Coagulation and Inflammation through Generation of Tissue Factor-Positive Microvesicles

Tissue factor (TF) is a cellular receptor for the clotting protease factor VIIa (FVIIa), and TF-FVIIa complex initiates activation of the coagulation cascade. TF-FVIIa also influences non-hemostatic functions via direct or indirect cell signaling. Constitutive expression of TF is primarily restricted to the surface of perivascular cells, sequestered from circulating blood clotting factors, to prevent aberrant intravascular clotting. It has been suggested that TF is also present in the blood associated with microvesicles (MVs). These circulating TF, however, do not induce any thrombotic complications as they are either cleared from the system before they could accumulate to a pathological level or lack procoagulant activity. However, various pathological conditions such as sepsis, diabetes, bacterial infection, atherosclerosis, and cancer can increase the levels of circulating TF+ MVs, which can then trigger intravascular coagulation and inflammation. We hypothesized that oxidative stress, a pathological condition, central to most of the diseases mentioned above, plays a role in the generation of TF+ MVs in vivo, which could contribute to intravascular coagulation and inflammation. We recently showed that 4-hydroxy-2-nonenal (HNE), the most abundant and stable unsaturated aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids, enhances TF activity in monocytic cells and macrophages. However, the role of HNE in activation of intravascular coagulation is unknown. To investigate this, C57Bl/6J wild-type mice were administered with HNE intraperitoneally (10 mg/kg body weight) and generation of TF+ MVs, activation of intravascular coagulation, and inflammation was assessed. HNE administration markedly increased TF activity associated with MVs. Nanoparticle tracking analysis (NTA) showed a 3-fold increase in the number of MVs in the plasma of HNE-treated mice. Measurement of thrombin-antithrombin (TAT) complexes revealed a significant increase in TAT levels in HNE treated mice. Pre-treatment of the mice with anti-murine TF antibody attenuated the HNE-induced increase in TAT levels. Measurement of plasma clotting times showed a significant reduction in HNE treated mice (118.3 ± 10.42 sec) compared to saline treated control mice (> 300 sec). Incubation of plasma with anti-TF antibody reversed the reduction observed in the plasma clotting time of HNE treated mice. Assessment of clotting in the saphenous vein injury bleeding model showed an increased number of clots, and reduced blood loss in HNE-administered mice compared to saline treated mice. Blockade of TF with 1H1 anti-TF antibody negated the HNE-mediated reduction in the clotting time. Next, we investigated whether HNE administration also induces a proinflammatory response in mice. Blood cells analysis showed approximately a 4-fold increase in circulating neutrophils in HNE-treated mice at 6 h following HNE treatment. Immunohistochemistry studies showed a robust infiltration of neutrophils into the lungs. Cytokine analysis of plasma showed a significant increase in IL-6 and CXCL1 levels in HNE-treated mice. Blockade of TF using 1H1 anti-TF antibody significantly reduced the HNE-mediated neutrophil infiltration into the lungs and completely abrogated the increase in plasma IL-6 levels. In additional studies, we attempted to identify the source of TF+ MVs. It is unlikely that they are originated from monocytes or endothelial cells as HNE did not induce TF expression in these cell types. LPS, used as a positive control, showed significant induction of TF on both PBMCs and the endothelium Immunohistochemistry revealed that HNE disrupted endothelial barrier integrity of vessel wall, raising a possibility that TF+ MVs could have come from perivascular cells. In summary, our data indicate that oxidative stress could induce the release of TF+ MVs, and this would contribute to intravascular coagulation and inflammation. Our studies also suggest that TF-positive microvesicles can be a useful therapeutic target in the treatment of thromboinflammatory diseases. Disclosures No relevant conflicts of interest to declare.

Vincristine induces procoagulant activity of the human lymphoblastic leukemia cell line Jurkat through the release of extracellular vesicles.

Thromboembolic events are frequent and serious complications of acute lymphoblastic leukaemia treatment. The importance of chemotherapy in the pathogenesis of this increased risk is enhanced by the fact that thrombosis rarely occurs at diagnosis. Our study aims at investigating the effect of chemotherapy on pro-coagulant activity (PCA), phosphatidylserine (PS) exposure, tissue factor (TF) activity and derived extracellular vesicles (EV) of Jurkat cells. Jurkat cells were treated with two commonly used chemotherapeutics: Vincristine (VCR) or Daunorubicin (DNR), at relevant concentrations. PCA of cells and derived EV were evaluated using Thrombin generation Assay (TGA). Cells or EV were incubated with annexin V or anti TF antibodies to assess the respective contribution of TF and PS. PS exposure on cells was analysed by flow cytometry. Derived EV were evaluated in fluorescence microscopy and flow cytometry. Untreated Jurkat cells and EV support thrombin generation. Thrombin generation was abolished when PS activity was inhibited by annexin V. VCR treatment resulted in a time dependent increase of thrombin generation. After VCR exposure, TF activity increased as well as PS exposure increased on the cell surface. The increase in TF activity was abolished by annexin V indicating that PS was required. A spontaneous release of EV from Jurkat cells was observed and VCR treatment increased the number of generated EV. Our results indicate that VCR increased the PCA of Jurkat cells predominantly through PS exposure and increased EV generation. Lymphoid blasts derived EV could be biomarkers to determine high thrombotic risk ALL patients.

Upregulation of Tissue Factor May Contribute to Thrombosis in Polycythemia Vera and Essential Thrombocythemia

Thrombosis is a major cause of morbidity and mortality in polycythemia vera (PV) and essential thrombocythemia (ET). The mechanistic basis of thrombosis in PV/ET, however, is unknown. To better understand the pathophysiology of thrombosis in PV and ET, we first studied transcript levels of selected thrombotic, inflammatory and hypoxia- inducible factor (HIF) pathway genes in granulocytes and platelets of PV and ET patients with and without thrombosis. Genes selected for the study included: tissue factor (F3); P-selectin (SELP); serpin peptidase inhibitor clade E member 1 (SERPINE1, encoding plasminogen activator inhibitor I, PAI1); thrombospondin 1 (THBS1); interleukin 1 receptor associated kinase 1 (IRAK1); interleukin 1 receptor accessory protein (IL1RAP) and HIF-regulated genes: vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (SLC2A1, encoding glucose transporter 1). We have previously reported at this meeting that PV and ET patients with a history of thrombosis had higher transcripts of F3, SERPINE1, IL1RAP, VEGFA and SLC2A1 compared to those without thrombosis. We also performed unbiased total RNA sequencing of platelets and granulocytes (Gangaraju R et al Blood, 2016;128:3143).Tissue factor (TF) is the principal initiator of coagulation in vivo. The presence of TF transcript in leukocytes and platelets may or may not reflect the translated protein level. Furthermore, TF functional activity is modulated by encryption. Therefore, we proceeded to evaluate the functional activity of microvesicle-associated TF (MVTF) in the plasma of 10 ET and 33 PV patients considered to have high thrombotic risk (Tefferi A, Barbui T Am J Hematol. 2017;92(1):94-108). TF activity was measured in MVs collected by centrifugation of patient plasma to 20,200 xg by a two-step FXa generation assay with and without an inhibitory TF antibody to determine the contribution of TF to FXa generation (Owens 3d et al. Circ Res. 2011;108(10):1284-97). We found significantly increased levels of MVTF activity in PV and ET compared to normal controls (Figure 1). However, MVTF levels in PV and ET patients with and without thrombosis were comparable (Figure 1).In the vasculature, leukocytes can synthesize TF (upon stimulation) and it has been shown that monocytes, not neutrophils, are the principal source of TF under normal conditions (Osterud B, Thromb Res. 2010;25 Suppl 1:S31-4). MPN granulocytes, in contrast to normal granulocytes, had increased levels of TF transcripts, a novel and important finding of as yet undetermined significance (Figure 2).Since TF synthesis is regulated by hypoxia-inducing factor-1 (HIF-1) (Rolfs et al. J Biol Chem. 1997;272(32):20055-62), we also examined MVTF activity in the plasma of Chuvash polycythemia (CP) patients. These patients have a germline VHLC598T mutation in the negative regulator of HIFs, the von Hippel Lindau gene (VHLC598T), and as a result they have increased levels of HIF-1, HIF-2 and transcripts of a vast array of HIF-regulated genes. CP subjects have an even higher propensity for arterial and venous thrombosis than PV. As predicted, some CP plasmas also demonstrated elevated levels of MVTF activity (Figure 1). In conclusion, hypoxia-induced increased levels of plasma MVTF activity may play a role in the increased thrombotic risk of PV and ET. TF joins TSP-1 (Sergeueva et al. Haematologica. 2017;102(5):e166-e169) and protein S (Pilly et al. Blood 2018;132(4):452-455) as a potential HIF-regulated mechanism of thrombotic risk in patients with PV/ET and CP. Granulocytes may also be a source of hypoxia-induced TF in these patients. The hypoxia-mediated upregulation of thrombotic risk is further underscored by the observation that PV patients living in moderate hypoxia at Salt Lake City have a higher risk of thrombosis than those living at sea level (Baltimore MD) in multivariate analysis (Zangari et al, Blood Coagul Fibrinolysis 2013; 24(3):311-316). The data described here may facilitate identification of novel targets and their therapies including the use of HIF-1 inhibitors such as digoxin to prevent thrombosis in these patients (Zhang et al. PNAS 2008;105(50):19579-86). [Display omitted] DisclosuresKey:UniQure BV: Research Funding.

Hazard characterization of an anti-human tissue factor antibody by combining results of tissue cross-reactivity studies and distribution of hemorrhagic lesions in monkey toxicity studies

Tissue cross-reactivity (TCR) studies are conducted when developing therapeutic antibodies, but their value is sometimes questioned because the positive organs often do not match the target organs of toxicity. We conducted TCR studies in human and cynomolgus monkey tissues for the development of an anti-human tissue factor antibody (TFAb) and also for a commercially available antibody, to clarify the true distribution of the target antigen. Tissue factor (TF) was found to be distributed in a wide variety of organs and tissues, including the heart and urinary bladder, in human and monkey. Administration of the TFAb to cynomolgus monkey caused hemorrhagic lesions mainly in the heart and urinary bladder in an incidental manner. This was thought to show the physiological role of TF in regulating hemostasis in these organs. Because the distribution of antigen in human and monkey was similar, the possibility that the TFAb would have similar effects in human was judged to be high, and because of the incidental nature of the effects, that they would be difficult to avoid. Thus it was possible to prospectively characterize the hazardous potential of a therapeutic antibody by accurately evaluating the tissue distribution of the target antigen and understanding its biological nature.

Role of Microparticles Derived from Acute Promyelocytic Leukemia Cells in Coagulopathy

To evaluate the role of microparticle (MP) derived from acute promyelocytic leukemia (APL) cells and tissue factor (TF) carried by the MP in hypercoagulable state, and the effect of treatment with cytotoxic chemotherapy/differentiating agents on procoagulant activity (PCA) of these MP. Bone marrow mononuclear cells (BMMNC) were extracted from 5 APL patients and 5 sex- and age- matched patients with iron deficiency anemia as controls. The cells were cultured in vitro for 48 h, then MP-rich culture medium and MP-free culture medium were harvested and MP was further obtained from certain volume of MP-rich culture medium. Subsequently, TF expression on MP was measured by ELISA. PCA of MP-rich culture medium or MP-free culture medium was assessed with thrombin generation assay. The role of TF on MP-related PCA was evaluated using anti-human TF antibody. In addition, APL cells were treated with all-trans retinoic acid (ATRA), arsenic trioxide (ATO) or daunorubicin (DNR) for 48 h, then MP-rich culture medium were harvested and the PCA was determined. No TF expression was found in the MP released from bone marrow MNC in control group, whereas the obvious TF expression was found in the MP originated from BMMNC of APL. MP from both APL and control BM-MNC had obvious PCA. However, compared with the MP derived from control MNC, the MP from APL BM-MNC induced significantly higher PCA. TF played a crucial role in the PCA of APL BM-MNC derived MP, while played no role in that of MP from control MNCs. DNR-treating APL BM-MNC resulted in an increase in the PCA of MP, whereas ATO or ATRA exposure lead to exactly the opposite results. MP derived from APL BM-MNC posseses obvious PCA. TF plays a crucial role in the MP-related PCA. The PCA of MP increases after treating APL BM-MNC with chemotherapy agent DNR and decreases following exposure of APL BM-MNC to differentiating agents ATRA or ATO.

Phosphatidylserine exposing-platelets and microparticles promote procoagulant activity in colon cancer patients.

BackgroundColon cancer is invariably accompanied by altered coagulation activity; however, the precise role of phosphatidylserine (PS) in the hypercoagulable state of colon cancer patients remains unclear. We explored the exposure of PS on platelets and microparticles (MPs), and evaluate its role in procoagulant activity in colon cancer patients.MethodsPS-positive platelets and MPs, mainly from platelets and endothelial cells, were detected by flow cytometry and confocal microscopy, and their procoagulant activity was assessed with purified coagulation complex assays, clotting time, and fibrin turbidity.ResultsPlasma levels of PS-positive platelets increased gradually from stage I to IV and were higher in all stages of the patients than in the healthy control, while PS-positive platelet-derived MPs only increased significantly in stage III/IV patients. Meanwhile, PS-positive MPs and endothelial-derived MPs in stage II/III/IV patients were markedly higher than ones in controls but no difference with stage I. Tissue factor positive MPs were higher in all 4 stages of colon cancer patients than in the healthy control. Platelets and MPs from the patients demonstrated significantly enhanced intrinsic/extrinsic FXa and thrombin generation, greatly shortened coagulation time, and increased fibrin formation. Combined treatment with PS antagonist lactadherin, strongly prolonged the coagulation time and reduced fibrin formation by inhibiting factor tenase and prothrombinase complex activity. In contrast, pretreatment with anti tissue factor antibody played a lesser role in suppression of procoagulant activity.ConclusionOur results suggest that PS-positive platelets and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with colon cancer.

Tissue factor associates with survival and regulates tumour progression in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumour. Patients often develop lung metastasis and have a poor prognosis despite extensive chemotherapy and surgical resections. Tissue Factor is associated with poor clinical outcome in a wide range of cancer types, and promotes angiogenesis and metastasis. The role of Tissue Factor in OS tumourigenesis is unknown. Fifty-three osteosarcoma pre-treatment biopsies and four osteosarcoma cell lines were evaluated for Tissue Factor expression, and a possible association with clinical parameters was investigated. Tissue Factor function was inhibited in an osteosarcoma cell line (143B) by shRNA knockdown or specific antibodies, and pro-tumourigenic gene expression, proliferation, matrigel invasion and transwell migration was examined. 143B cells were implanted in mice in the presence of Tissue Factor-blocking antibodies, and tumour volume, micro-vessel density and metastases in the lung were evaluated. Tissue Factor was highly expressed in 73.6 % of osteosarcoma biopsies, and expression associated significantly with disease-free survival. Tissue Factor was expressed in all four investigated cell lines. Tissue Factor was knocked down in 143B cells, which led to reduced expression of IL-8, CXCL-1, SNAIL and MMP2, but not MMP9. Tissue Factor knockdown or inhibition with antibodies reduced matrigel invasion. Tissue Factor antibodies limited 143B tumour growth in vivo, and resulted in decreased intra-tumoural micro-vessel density. Furthermore, lung metastasis from the primary tumour was significantly reduced. Thus, Tissue Factor expression in osteosarcoma reduces metastasis-free survival in patients, and increases pro-tumourigenic behaviour both in vitro and in vivo.

Platelet-Derived Beta Thromboglobulin: A Potential Novel Activator of Coagulation Factor X

BackgroundDuring clotting, factors IXa and VIIa participate in the activation of Factor X, leading to thrombin generation. This process occurs on the surface of activated platelets. Activated platelets release an array of proteins that can modulate thrombin generation. This includes platelet factor 4 (PF4), a chemokine that modulates coagulation by promoting protein C activation and by attenuating the anticoagulant activity of activated protein C. PF4 shares significant homology with another protein released in high concentration by platelets, β-thromboglobulin (βTG). The physiological effects of βTG, including effects on blood coagulation, are poorly characterized. The aim of this study was to assess the effect of βTG on blood coagulation.MethodsThe effect of purified human βTG on coagulation was determined using the prothrombin time (PT), activated partial thromboplastin time (APTT), calibrated automated thrombography (4µM phospholipids ± 1pM tissue factor (TF)) and chromogenic FX and prothrombin activation assays. Mass spectrometry and surface plasmon resonance (SPR) were used to assess the composition of the purified βTG preparation and to measure protein-protein interactions respectively.ResultsMass spectrometry confirmed the absence of TF, FVIIa, FVIIIa, FIXa, FVa, and FXa in the βTG preparation. In normal pooled plasma, βTG dose-dependently increased the rate and extent of TF stimulated thrombin generation. In the absence of βTG, the lagtime was 9±1 min, which was shortened in a dose-dependent manner upon incubation with βTG (100µg/ml; 5±1 min, p<0.05). Incubation with βTG (50µg/ml) also shortened the APTT (35±1 to 25±1 secs, p<0.05) and reduced the lagtime to thrombin generation (26±5 to 7±1 min, p<0.05) and increased the peak thrombin generation (60±30 to 97±23nM, p<0.05) in the absence of an exogenous TF stimulus. Immunodepleted plasmas and inhibitory antibodies were used to determine the underlying mechanism of action. In FVIII-deficient plasma, thrombin generation was not observed in the absence of an exogenous TF stimulus. However, upon incubation with βTG (50µg/ml), thrombin generation was observed and peak thrombin generation increased from 1±1 to 75±12nM IIa (p<0.05). βTG also shortened the APTT in FVIII-deficient plasma from 131±8 to 103±14 secs (50µg/ml, p<0.05). The procoagulant effect of βTG in FVIII-deficient plasma was not inhibited by TF or FVIIa inhibitory antibodies, suggesting that the effect was independent of the intrinsic tenase complex, TF or FVIIa. Interestingly, homologous PF4 did not induce thrombin generation in FVIII-deficient plasma (peak thrombin generation was 75±12nM v 0±0nM IIa upon incubation with 50µg/ml βTG and PF4 respectively). The procoagulant effect of βTG persisted when thrombin generation was independent of FV activation (supplementation of FV-deficient plasma with FVa). In contrast, the effect was not observed when thrombin generation was independent of FX activation (supplementation of FX-deficient plasma with FXa). Collectively, these data raised the possibility that βTG may modulate FX activation. To investigate this hypothesis, chromogenic FX activation was measured. Cleavage of a FXa-specific chromogenic substrate was observed upon incubation of βTG with FX, suggesting a direct effect of βTG upon FX activation. No measurable chromogenic substrate cleavage was observed upon incubation with βTG or FX alone. In contrast, βTG did not induce prothrombin activation, measured using a thrombin-specific chromogenic substrate. Using SPR, βTG was found to bind directly to immobilised FX (KD 1.36±0.7x10-7 M). The kinetics of the interaction between βTG and FX was lower than that between FIXa and FX (KD 4.25±1.90 × 10-9 M).ConclusionIn conclusion, we have identified that βTG is a potential novel platelet-derived activator of coagulation FX. Moreover, βTG is capable of inducing thrombin generation in immunodepleted FVIII and FIX-deficient plasma, a finding that may be of translational relevance to patients with haemophilia. DisclosuresNí Áinle:Actelion Pharma: Research Funding; Leo Pharma: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

Abstract 398: Inhibition of Tissue Factor Reduces Thrombosis and Inflammation Without Increased Bleeding in a Mouse Model of Ischemic Stroke

Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, is highly expressed by astrocytes surrounding blood vessels in the central nervous system. Using mouse models of hemorrhagic and ischemic stroke, we previously demonstrated that astrocyte TF plays a critical role in brain hemostasis while also contributing to neuronal damage. Importantly, using mice with different levels of astrocyte TF expression, we determined that an astrocyte TF activity around 5% of the wild type level was sufficient to limit bleeding in hemorrhagic stroke and at the same time was low enough to reduce infarct size and behavioral deficits in ischemic stroke, without increased hemorrhagic transformation. Therefore, we hypothesized that pharmacologic inhibition of TF may improve outcomes after ischemic stroke and have a wide safety margin. To test this hypothesis, C56Bl/6J mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hour followed by 24 hours of reperfusion. Inhibitory rat anti- mouse TF antibody (1H1) and control IgG were administered as a bolus intraperitoneal injection immediately after ischemia. Inhibition of TF dose-dependently reduced brain infarct size in female mice (36.6±1.4% of total brain volume in IgG group [n=19] vs 28.3 ± 1.7% in 25mg/kg 1H1 group [n=9, p&lt;0.01] and 18.5 ±2.3% in 75mg/kg 1H1 group [n=8, p&lt;0.001]; mean±SEM). Importantly, 1H1 did not exacerbate hemorrhagic transformation during the reperfusion phase. Furthermore, TF inhibition significantly reduced microvascular thrombosis (analyzed by fibrin staining), expression of inflammatory markers in the injured brain (IL-6, MCP-1 and KC levels- analyzed by ELISA) and attenuated behavioral deficit after ischemic stroke. Subjecting male mice to the same MCAO condition resulted in partial lethality in IgG treated group (4 out of 6 mice died with 24 hours) which was prevented by 1H1 (25mg/kg) treatment (0 out of 5, Log-rank test p=0.031). Altogether, our data suggest that the overabundance of astrocyte TF expression resulted from selective pressure to limit intracerebral hemorrhage after traumatic brain injury, but in the modern era poses the additional risk of increased injury during ischemic stroke.

Equid herpesvirus type 1 activates platelets.

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.

Antitumor effect of antitissue factor antibody-MMAE conjugate in human pancreatic tumor xenografts.

Tissue factor (TF) triggers the extrinsic blood coagulation cascade and is highly expressed in various types of cancer. In this study, we investigated the antitumor effect of an antibody–drug conjugate (ADC) consisting of an anti‐TF monoclonal antibody and monomethyl auristatin E (MMAE). MMAE was conjugated to an anti‐human TF or anti‐mouse TF antibody using a valine‐citrulline linker that could be potentially hydrolyzed by cathepsin B in the acidic environment of the lysosome. The cytotoxic and antitumor effects of the ADCs against four pancreatic cancer cell lines were analyzed. Both the ADC with the anti‐human TF antibody and that with the anti‐mouse TF antibody were stable under physiological conditions. The anti‐human ADC was internalized in TF‐expressing human tumor cell lines, followed by effective MMAE release. The half maximal inhibitory concentration (IC50) of MMAE was approximately 1 nM for all of the cell lines used. Meanwhile, the IC50 of anti‐human ADC was 1.15 nM in the cell lines showing high TF expression, while exceeding 100 nM in the cells showing low TF expression levels. Anti‐human ADC with passive and active targeting ability exerted significant suppression of tumor growth as compared to that observed in the saline group (p < 0.01). Also significant tumor growth suppressions were seen at the anti‐mouse ADC and control ADC groups compared to the saline group (p < 0.01) due to EPR effect. Because various clinical human cancers express highly amount of TF, this new anti‐TF ADC may deserve a clinical evaluation.

Tissue factor and atherothrombosis.

Tissue factor (TF) is known to be the key element in the initiation of the extrinsic pathway of the coagulation cascade and appears to be a critical determinant of atherosclerotic plaque thrombogenicity. TF is needed to produce thrombin from prothrombin. In the extrinsic pathway, TF activates factor Vll. TF is expressed mainly on subendothelial tissues, but TF expression may be induced on endothelial cells by inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha). Subendothelial TF is responsible for initiating fibrin formation at sites of vascular injury, bloodborne TF may be an important contributor to propagation of the developing thrombus. It has been postulated that the blood-borne TF initiates the thrombogenic stimulus, leading to the formation of larger and more stable thrombus. TF may attach to cellular receptors, which in turn affect the production and release of inflammatory mediators. Baseline plasma TF activity has been demonstrated as an independent predictor for cardiovascular death in patients with acute myocardial infarction. TF is expressed by macrophage-derived foam cells in atherosclerotic plaques. TF levels were higher in atheroma from patients with unstable angina than with stable angina. These results suggest that high levels of TF exposed upon plaque rupture trigger atherothrombosis. Inhibition of TF would be expected to reduce thrombosis associated with a variety of diseases. TF pathway is a potential target for new therapeutic agents that can decrease TF activity, such as active site-inactivated factor VIIa, recombinant TF inhibitor and antibodies against TF or peptides interfering with TF-FVIIa complex activity. Significant clinical forms of atherosclerosis, such as sudden death, myocardial infarction, and stroke have common pathogenesis. The occlusion of the vessel lumen is the result from atherosclerotic plaque rupture/erosion that initiate thrombus formation. This thrombus has complex structure and contains predominantly fibrin in addition to platelets, suggesting an important role for the coagulation cascade in plaque thrombus formation. Tissue factor (TF) is known to be the key element in the initiation of the extrinsic pathway of the coagulation cascade and appears to be a critical determinant of atherosclerotic plaque thrombogenicity.

Abstract 2642: Antibody-drug conjugate for human pancreatic cancer cells using anti-tissue factor monoclonal antibody

Abstract Background: The incidence of venous thromboembolism in patients with cancer is reported to be twofold higher than that in patients without cancer. One of the well-characterized procoagulants associated with cancer is tissue factor (TF). TF is expressed not only on the surfaces of the cancer cells, but also in the monocytes and endothelial cells which are stimulated under the cancerous condition. Thus, TF is an appropriate molecule for drug delivery to both cancer cells and cancer stroma. In the present study, the antitumor effect of an antibody drug conjugate (ADC), anti-TF monoclonal antibody (mAb) conjugated with monomethyl auristatin E (MMAE), was investigated. Methods: MMAE was conjugated to anti-human TF (hTF) and -mouse TF (mTF) antibody using a valine-citrulline dipeptide linker. The stability of ADC under the physiological and intracellular conditions were analyzed by HPLC. The bio-distributions of hTF and mTF mAbs were analyzed in vivo. The cytotoxic effects of the ADC in four pancreatic cancer cell lines were analyzed both in vitro and in vivo. Results: The four pancreatic cancer cell lines used in this study were classified into 3 groups; high-TF-expressing cells (BxPC-3), moderate-TF-expressing cells (PSN-1), and low-TF-expressing cells (Capan-1 and Panc-1). Anti-hTF mAb was sufficiently internalized in the cytoplasm of BxPC-3 after 3-h incubation. The ADC was stable under the extracellular condition, and sufficient free MMAE was released into the cytoplasm. hTF mAb permeating from the tumor vessels was distributed at the periphery of the tumors. On the other hand, mTF mAb was distributed in the cancer stroma. The IC50 of MMAE for these pancreatic cancer cell lines was approximately 1 nM, while the IC50 of the MMAE contained in hTF-MMAE for BxPC-3 was 1.15 nM; on the other hand the values for Capan-1 and Panc-1 exceeded 100 nM. BxPC-3 subcutaneous tumor growth was significantly suppressed in the 20 mg/kg hTF-MMAE group as compared with that in the saline group (P &amp;lt; 0.001). Tumor growth in the 20 mg/kg mTF-MMAE group was also significantly suppressed as compared with that in the saline group (P = 0.001). The Ki67-positive cell rate in the hTF-MMAE treatment group was significantly lower than that in the saline (P = 0.001) and mTF-MMAE treatment groups (P = 0.015). Conclusions: ADC measuring 10 nm in size could reach cancer cells through the cancer stroma and could be effectively internalized into cancer cells. Meanwhile, cancer-stroma-targeting mAb (mTF mAb) elicit a partial response as compared to hTF mAb. Our anti-TF mAb conjugated with MMAE warrants clinical evaluation. Citation Format: Yoshikatsu Koga, Ryuta Sato, Ryo Tsumura, Hikaru Machida, Yoshiyuki Yamamoto, Yohei Hisada, Yuki Fujiwara, Masahiro Yasunaga, Shino Manabe, Yasuhiro Matsumura. Antibody-drug conjugate for human pancreatic cancer cells using anti-tissue factor monoclonal antibody. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2014-2642

Roles of Coagulation and Fibrinolysis in Angiotensin II‐Enhanced Microvascular Thrombosis

AngII-induced HTN is associated with accelerated thrombus development in arterioles. This study assessed the contributions of different components of the coagulation cascade and fibrinolysis to AngII-mediated microvascular thrombosis. Light/dye-induced thrombus formation (the time of onset and flow cessation) was quantified in cremaster muscle arterioles of AngII infused (two weeks) WT/AngII mice, EPCR-TgN, and mice deficient in PAI-1. WT/AngII mice were also treated with either tissue factor antibody, antithrombin III, heparin, hirudin, or murine APC. TF immunoblockade or hirudin treatment did not prevent the AngII-induced acceleration of thrombosis. While antithrombin III treatment prevented the acceleration in both thrombus onset and flow cessation, heparin only improved the time for blood flow cessation. Neither WT mice treated with murine APC nor EPCR-TgN were protected against AngII-induced thrombus development. A similar lack of protection was noted in PAI-1deficient mice. These findings implicate a role for thrombin generation pathway in the accelerated thrombosis induced by AngII and suggest that an impaired protein C pathway and increased PAI-1 do not make a significant contribution to this model of microvascular thrombosis.